WO2010032138A2 - Combinaisons d’adjuvant de vaccin - Google Patents

Combinaisons d’adjuvant de vaccin Download PDF

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Publication number
WO2010032138A2
WO2010032138A2 PCT/IB2009/007111 IB2009007111W WO2010032138A2 WO 2010032138 A2 WO2010032138 A2 WO 2010032138A2 IB 2009007111 W IB2009007111 W IB 2009007111W WO 2010032138 A2 WO2010032138 A2 WO 2010032138A2
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Prior art keywords
adjuvant
oil
emulsion
immunogen
average diameter
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PCT/IB2009/007111
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English (en)
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WO2010032138A3 (fr
Inventor
Michele Pallaoro
Derek O'hagan
Rino Rappuoli
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Novartis Ag
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Priority to CN2009801371805A priority Critical patent/CN102159242A/zh
Priority to AU2009294318A priority patent/AU2009294318B2/en
Priority to US13/119,917 priority patent/US20110236489A1/en
Priority to JP2011527427A priority patent/JP2012502972A/ja
Priority to NZ591768A priority patent/NZ591768A/xx
Priority to EP09748472A priority patent/EP2331127A2/fr
Priority to CA2737455A priority patent/CA2737455A1/fr
Publication of WO2010032138A2 publication Critical patent/WO2010032138A2/fr
Publication of WO2010032138A3 publication Critical patent/WO2010032138A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59

Definitions

  • This invention is in the field of vaccine adjuvants and their combinations.
  • Oil-in-water emulsions are known for use as vaccine adjuvants, and the FLU ADTM product includes the squalene-in-water 'MF59' adjuvant. It is an object of the invention to provide modified and improved emulsion adjuvants.
  • the invention provides an immunological adjuvant comprising an oil-in-water emulsion, an immunostimulatory oligonucleotide and a polycationic polymer.
  • the oligonucleotide and the polymer ideally associate with each other to form a complex.
  • the invention also provides a process for preparing an immunological adjuvant of the invention, comprising a step of mixing an oil-in-water emulsion with a complex of an immunostimulatory oligonucleotide and a polycationic polymer.
  • the invention also provides an immunogenic composition comprising (i) an adjuvant of the invention and (ii) an immunogen.
  • the invention also provides a process for preparing an immunogenic composition comprising a step of mixing (i) an adjuvant of the invention and (ii) an immunogen.
  • the immunogen, emulsion, oligonucleotide and polymer may be mixed in any order.
  • the invention provides a process for preparing an immunogenic composition of the invention, comprising a step of mixing (i) an oil-in-water emulsion and (ii) an immunogen; and then mixing the emulsion/immunogen mixture with an immunostimulatory oligonucleotide and a polycationic polymer.
  • the invention also provides a process for preparing an immunogenic composition of the invention, comprising a step of mixing (i) an immunostimulatory oligonucleotide and a polycationic polymer, typically in the form of a complex, and (ii) an immunogen; and then mixing the oligonucleotide/polymer/immunogen mixture with an oil-in-water emulsion.
  • the invention also provides a kit comprising: (i) a first container that contains an adjuvant of the invention; and (ii) a second container that contains an immunogen and/or a further adjuvant.
  • the invention also provides a kit comprising: (i) a first container that contains an oil-in-water emulsion; and (ii) a second container that contains an immunostimulatory oligonucleotide and a polycationic polymer.
  • a kit comprising: (i) a first container that contains an oil-in-water emulsion; and (ii) a second container that contains an immunostimulatory oligonucleotide and a polycationic polymer.
  • One or both of the first and second containers may include an immunogen.
  • the contents of the two containers can be combined (e.g. at the point of use) to form an adjuvant or immunogenic composition of the invention.
  • kits may include a third container that contains an immunogen and/or a further adjuvant.
  • the invention also provides an immunological adjuvant comprising an oil-in-water emulsion and an adsorptive particulate adjuvant, wherein the average diameter of particles in the adsorptive particulate adjuvant and the average diameter of oil droplets in the emulsion are both less than 250nm (e.g. ⁇ 220nm, ⁇ 200nm, ⁇ 190nm, ⁇ 180nm, ⁇ 150nm, ⁇ 120nm, ⁇ 100nm,e/c). If the particulate adjuvant has particles with a range of diameters then these diameters may not overlap with the oil droplet particles sizes (i.e.
  • the largest oil droplets are smaller than the smallest adjuvant particles, or the largest adjuvant particles are smaller than the smallest oil droplets). In other embodiments, however, the size distributions may overlap. In some embodiments the average diameter of the adjuvant particles may be substantially the same as the average diameter of the oil droplets, or these two diameters may differ e.g. by at least 5%, 10%, 15%, 20%, 25%, etc.
  • the invention also provides an immunological adjuvant comprising an oil-in-water emulsion and an adsorptive particulate adjuvant, wherein the average diameter of particles in the adsorptive particulate adjuvant is greater than the average diameter of oil droplets in the emulsion.
  • the adsorptive particulate adjuvant ideally does not comprise (i) an insoluble aluminium or calcium salt or (ii) polymeric microparticles, but rather is preferably (iii) a complex of an immunostimulatory oligonucleotide and a polycationic polymer. Mixing an oil-in-water emulsion with such complexes is shown herein to reduce the complexes' analysed mean diameter.
  • the invention also provides an immunogenic composition
  • an immunogenic composition comprising (i) an immunological adjuvant comprising an oil-in-water emulsion and an adsorptive particulate adjuvant; and (ii) an immunogen.
  • the adsorptive particulate adjuvant ideally does not comprise an insoluble aluminium or calcium salt or a polymeric microparticle but is preferably a complex of an immunostimulatory oligonucleotide and a polycationic polymer.
  • the immunogen is at least partially adsorbed to the adsorptive particulate adjuvant.
  • the invention also provides an immunological adjuvant comprising an oil-in-water emulsion and an immunostimulatory oligonucleotide, wherein the immunostimulatory oligonucleotide includes at least one CpI motif (a dinucleotide sequence containing a cytosine linked to an inosine).
  • the oligodeoxynucleotide may include more than one (e.g. 2, 3, 4, 5, 6 or more) CpI motif, and these may be directly repeated (e.g. comprising the sequence (CI) * , where x is 2, 3, 4, 5, 6 or more) or separated from each other (e.g.
  • the invention also provides a process for preparing this immunological adjuvant, comprising a step of mixing an oil-in-water emulsion with a Cpl-containing immunostimulatory oligonucleotide.
  • the invention also provides an immunogenic composition comprising (i) this adjuvant and (ii) an immunogen.
  • the invention also provides a process for preparing an immunogenic composition comprising a step of mixing (i) this adjuvant and (ii) an immunogen.
  • the invention also provides a process for preparing an immunogenic composition of the invention, comprising a step of mixing (i) an oil-in-water emulsion and (ii) an immunogen; and then mixing the emulsion/immunogen mixture with a Cpl-containing immunostimulatory oligonucleotide.
  • the invention also provides a process for preparing an immunogenic composition of the invention, comprising a step of mixing (i) a Cpl-containing immunostimulatory oligonucleotide and (ii) an immunogen; and then mixing the oligonucleotide/immunogen mixture with an oil-in-water emulsion.
  • Oil-in-water emulsions used with the invention typically include at least one oil and at least one surfactant, with the oil(s) and surfactant(s) being biodegradable (metabolisable) and biocompatible.
  • the oil droplets in the emulsion are generally less than 5 ⁇ m in diameter, and ideally have a sub-micron diameter, with these small sizes being achieved with a microfiuidiser to provide stable emulsions. Droplets with a size less than 220nm are preferred as they can be subjected to filter sterilization. In some useful emulsions at least 80% (by number) of the oil droplets have a diameter less than 500nm
  • the emulsions can include oils such as those from an animal (such as fish) or vegetable source.
  • Sources for vegetable oils include nuts, seeds and grains. Peanut oil, soybean oil, coconut oil, and olive oil, the most commonly available, exemplify the nut oils. Jojoba oil can be used e.g. obtained from the jojoba bean. Seed oils include safflower oil, cottonseed oil, sunflower seed oil, sesame seed oil, etc. In the grain group, corn oil is the most readily available, but the oil of other cereal grains such as wheat, oats, rye, rice, teff, triticale, etc. may also be used.
  • 6-10 carbon fatty acid esters of glycerol and 1 ,2-propanediol may be prepared by hydrolysis, separation and esterification of the appropriate materials starting from the nut and seed oils.
  • Fats and oils from mammalian milk are metabolizable and may therefore be used in the practice of this invention.
  • the procedures for separation, purification, saponification and other means necessary for obtaining pure oils from animal sources are well known in the art.
  • Most fish contain metabolizable oils which may be readily recovered. For example, cod liver oil, shark liver oils, and whale oil such as spermaceti exemplify several of the fish oils which may be used herein.
  • branched chain oils are synthesized biochemically in 5-carbon isoprene units and are generally referred to as terpenoids.
  • Shark liver oil contains a branched, unsaturated terpenoid known as squalene, 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene.
  • Squalane the saturated analog to squalene
  • Fish oils, including squalene and squalane are readily available from commercial sources or may be obtained by methods known in the art. Squalene is preferred.
  • tocopherols are advantageously included in vaccines for use in elderly subjects (e.g. aged 60 years or older) because vitamin E has been reported to have a positive effect on the immune response in this subject group. They also have antioxidant properties that may help to stabilize emulsions.
  • Various tocopherols exist ( ⁇ , ⁇ , ⁇ , ⁇ , ⁇ or ⁇ ) but ⁇ is usually used.
  • a preferred ⁇ -tocopherol is DL- ⁇ -tocopherol.
  • ⁇ -tocopherol succinate is known to be compatible with influenza vaccines and to be a useful preservative as an alternative to mercurial compounds.
  • oils can be used e.g. squalene and ⁇ -tocopherol.
  • An oil content in the range of 2-20% (by volume) is typical.
  • Surfactants can be classified by their 'HLB' (hydrophile/lipophile balance). Some surfactants useful with the invention have a HLB of at least 10 e.g. at least 15 or at least 16.
  • the invention can be used with surfactants including, but not limited to: the polyoxyethylene sorbitan esters surfactants (commonly referred to as the Tweens), especially polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO), sold under the DO WF AXTM tradename, such as linear EO/PO block copolymers; octoxynols, which can vary in the number of repeating ethoxy (oxy-l,2-ethanediyl) groups, with octoxynol-9 (Triton X-IOO, or t-octylphenoxypolyethoxyethanol) being of particular interest; (octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipids such as phosphatidylcholine (lecithin); nonylphenol ethoxylates, such as the TergitolTM
  • surfactants can be used e.g. Tween 80/Span 85 mixtures.
  • a combination of a polyoxyethylene sorbitan ester and an octoxynol is also suitable.
  • Another useful combination comprises laureth 9 plus a polyoxyethylene sorbitan ester and/or an octoxynol.
  • surfactants are: polyoxyethylene sorbitan esters (such as Tween 80) 0.01 to 1%, in particular about 0.1%; octyl- or nonylphenoxy polyoxyethanols (such as Triton X-100, or other detergents in the Triton series) 0.001 to 0.1 %, in particular 0.005 to 0.02%; polyoxyethylene ethers (such as laureth 9) 0.1 to 20 %, e.g. 0.1 to 10 % and in particular 0.1 to 1 % or about 0.5%.
  • polyoxyethylene sorbitan esters such as Tween 80
  • octyl- or nonylphenoxy polyoxyethanols such as Triton X-100, or other detergents in the Triton series
  • polyoxyethylene ethers such as laureth 9
  • Squalene-containing emulsions are preferred, particularly those containing polysorbate 80.
  • Specific oil-in-water emulsion adjuvants useful with the invention include, but are not limited to:
  • a submicron emulsion of squalene, polysorbate 80, and sorbitan trioleate The composition of the emulsion by volume can be about 5% squalene, about 0.5% polysorbate 80 and about 0.5% Span 85. In weight terms, these ratios become 4.3% squalene, 0.5% polysorbate 80 and 0.48%
  • MF59' This adjuvant is known as 'MF59' [1-3], as described in more detail in Chapter 10 of ref. 4 and chapter 12 of ref. 5.
  • the MF59 emulsion advantageously includes citrate ions e.g. 1OmM sodium citrate buffer.
  • a submicron emulsion of squalene, a tocopherol, and polysorbate 80 may have from 2 to 10% squalene, from 2 to 10% tocopherol and from 0.3 to 3% polysorbate 80, and the weight ratio of squalene:tocopherol is preferably ⁇ 1 (e.g. 0.90) as this can provide a more stable emulsion.
  • Squalene and polysorbate 80 may be present at a volume ratio of about 5:2 or at a weight ratio of about 1 1 :5.
  • One such emulsion can be made by dissolving Tween 80 in PBS to give a 2% solution, then mixing 90ml of this solution with a mixture of (5g of DL- ⁇ -tocopherol and 5ml squalene), then microfluidising the mixture.
  • the resulting emulsion has submicron oil droplets e.g. with an average diameter of between 100 and 250nm, preferably about 180nm.
  • the emulsion may also include a 3-de-O-acylated monophosphoryl lipid A (3d-MPL).
  • Another useful emulsion of this type may comprise, per human dose, 0.5-10 mg squalene, 0.5-11 mg tocopherol, and 0.1-4 mg polysorbate 80 [6].
  • An emulsion of squalene, a tocopherol, and a Triton detergent e.g. Triton X-100
  • the emulsion may also include a 3d-MPL (see below).
  • the emulsion may contain a phosphate buffer.
  • An emulsion comprising a polysorbate (e.g. polysorbate 80), a Triton detergent (e.g. Triton X-100) and a tocopherol (e.g. an ⁇ -tocopherol succinate).
  • the emulsion may include these three components at a mass ratio of about 75: 1 1 : 10 (e.g. 750 ⁇ g/ml polysorbate 80, HO ⁇ g/ml Triton X-100 and lOO ⁇ g/ml ⁇ -tocopherol succinate), and these concentrations should include any contribution of these components from antigens.
  • the emulsion may also include squalene.
  • the emulsion may also include a 3d-MPL.
  • the aqueous phase may contain a phosphate buffer.
  • An emulsion of squalane, polysorbate 80 and poloxamer 401 ("PluronicTM Ll 21").
  • the emulsion can be formulated in phosphate buffered saline, pH 7.4. This emulsion is a useful delivery vehicle for muramyl dipeptides, and has been used with threonyl-MDP in the
  • SAF-I adjuvant [7] (0.05-1% Thr-MDP, 5% squalane, 2.5% Pluronic L121 and 0.2% polysorbate 80). It can also be used without the Thr-MDP, as in the "AF" adjuvant [8] (5% squalane, 1.25% Pluronic L121 and 0.2% polysorbate 80). Microfluidisation is preferred.
  • An emulsion comprising squalene, an aqueous solvent, a polyoxyethylene alkyl ether hydrophilic nonionic surfactant (e.g. polyoxyethylene (12) cetostearyl ether) and a hydrophobic nonionic surfactant (e.g. a sorbitan ester or mannide ester, such as sorbitan monoleate or 'Span 80').
  • the emulsion is preferably thermoreversible and/or has at least 90% of the oil droplets (by volume) with a size less than 200 nm [9].
  • the emulsion may also include one or more of: alditol; a cryoprotective agent (e.g.
  • the emulsion may include a TLR4 agonist [10]. Such emulsions may be lyophilized.
  • An emulsion having from 0.5-50% of an oil, 0.1-10% of a phospholipid, and 0.05-5% of a non-ionic surfactant.
  • preferred phospholipid components are phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, sphingomyelin and cardiolipin. Submicron droplet sizes are advantageous.
  • Additives may be included, such as QuilA saponin, cholesterol, a saponin-lipophile conjugate (such as GPI-0100, described in reference 13, produced by addition of aliphatic amine to desacylsaponin via the carboxyl group of glucuronic acid), dimethyidioctadecylammonium bromide and/or N,N-dioctadecyl-N,N-bis (2-hydroxyethyl)propanediamine.
  • An emulsion in which a saponin (e.g. QuilA or QS21) and a sterol (e.g. a cholesterol) are associated as helical micelles [14].
  • An emulsion comprising a mineral oil, a non-ionic lipophilic ethoxylated fatty alcohol, and a non-ionic hydrophilic surfactant (e.g. an ethoxylated fatty alcohol and/or polyoxyethylene- polyoxypropylene block copolymer) [15].
  • An emulsion comprising a mineral oil, a non-ionic hydrophilic ethoxylated fatty alcohol, and a non-ionic lipophilic surfactant (e.g. an ethoxylated fatty alcohol and/or polyoxyethylene- polyoxypropylene block copolymer) [15].
  • the squalene concentration in a vaccine dose may be in the range of 5-15mg (i.e. a concentration of 10-30mg/ml, assuming a 0.5ml dose volume). It is possible, though, to reduce the concentration of squalene [ 16,17] e.g. to include ⁇ 5mg per dose, or even ⁇ l . lmg per dose.
  • a human dose may include 9.75mg squalene per dose (as in the FLUADTM product: 9.75mg squalene, 1.175mg polysorbate 80, 1.175mg sorbitan trioleate, in a 0.5ml dose volume), or it may include a fractional amount thereof e.g. 3/4, 2/3, 1/2, 2/5, 1/3, 1/4, 1/5, 1/6, 1/7, 1/8, 1/9, or 1/10.
  • a composition may include 4.875 squalene per dose (and thus 0.588mg each of polysorbate 80 and sorbitan trioleate), 3.25mg squalene/dose, 2.438mg/dose, 1.95mg/dose, 0.975mg/dose, etc. Any of these fractional dilutions of the FLUADTM-strength MF59 can be used with the invention, while maintaining a squalene:polysorbate-80:sorbitan-trioleate ratio of 8.3: 1 : 1 (by mass).
  • the immunostimulatory oligonucleotide and the polycationic polymer uses an immunostimulatory oligonucleotide and a polycationic polymer. These are ideally associated with each other to form a particulate complex, which usefully is a TLR9 agonist.
  • Immunostimulatory oligonucleotides are known as useful adjuvants. They often contain a CpG motif (a dinucleotide sequence containing an unmethylated cytosine linked to a guanosine) and their adjuvant effect is discussed in refs. 18-23. Oligonucleotides containing TpG motifs, palindromic sequences, multiple consecutive thymidine nucleotides (e.g. TTTT), multiple consecutive cytosine nucleotides (e.g. CCCC) or poly(dG) sequences are also known immunostimulants, as are double-stranded RNAs.
  • CpG motif a dinucleotide sequence containing an unmethylated cytosine linked to a guanosine
  • Inosine-containing oligodeoxynucleotides may include a CpI motif (a dinucleotide sequence containing a cytosine linked to an inosine).
  • the oligodeoxynucleotide may include more than one (e.g. 2, 3, 4, 5, 6 or more) CpI motif, and these may be directly repeated (e.g.
  • oligonucleotides comprising the sequence (CI) x , where x is 2, 3, 4, 5, 6 or more) or separated from each other (e.g. comprising the sequence (CIN) x , where x is 2, 3, 4, 5, 6 or more, and where each N independently represents one or more nucleotides). Cytosine residues are ideally unmethylated.
  • the oligonucleotides will typically have between 10 and 100 nucleotides e.g. 15-50 nucleotides, 20-30 nucleotides, or 25-28 nucleotides. It will typically be single-stranded.
  • the oligonucleotide can include exclusively natural nucleotides, exclusively non-natural nucleotides, or a mix of both. For instance, it may include one or more phosphorothioate linkage(s), and/or one or more nucleotides may have a 2'-O-methyl modification.
  • a preferred oligonucleotide for use with the invention is a single-stranded.deoxynucleotide comprising the 26-mer sequence 5'-(IC)i 3 -3' (SEQ ID NO: 1). This oligodeoxynucleotide forms stable complexes with polycatior ⁇ c polymers to give a good adjuvant.
  • the polycationic polymer is ideally a polycationic peptide.
  • the polymer may include one or more leucine amino acid residue(s) and/or one or more lysine amino acid residue(s).
  • the polymer may include one or more arginine amino acid residue(s). It may include at least one direct repeat of one of these amino acids e.g. one or more Leu-Leu dipeptide sequence(s), one or more Lys-Lys dipeptide sequence(s), or one or more Arg-Arg dipeptide sequence(s). It may include at least one (and preferably multiple e.g. 2 or 3) Lys-Leu dipeptide sequence(s) and/or at least one (and preferably multiple e.g.
  • the peptide may comprise a sequence R]-XZXZ x XZX-R 2 , wherein: x is 3, 4, 5, 6 or 7; each X is independently a positively-charged natural and/or non-natural amino acid residue; each Z is independently an amino acid residue L, V, I, F or W; and R
  • X-R 2 may be an amide, ester or thioester of the peptide's C-terminal amino acid residue.
  • a polycationic peptide will typically have between 5 and 50 amino acids e.g. 6-20 amino acids, 7-15 amino acids, or 9-12 amino acids.
  • a peptide can include exclusively natural amino acids, exclusively non-natural amino acids, or a mix of both. It may include L-amino acids and/or D-amino acids. L-amino acids are typical.
  • a peptide can have a natural N-terminus (NH 2 -) or a modified N-terminus e.g. a hydroxyl, acetyl, etc.
  • a peptide can have a natural C-terminus (-COOH) or a modified C-terminus e.g. a hydroxyl, an acetyl, etc. Such modifications can improve the peptide's sstability.
  • a preferred peptide for use with the invention is the 1 1-mer KLKLLLLLKLK (SEQ ID NO: 2), with all L-amino acids.
  • the N-terminus may be deaminated and the C-terminus may be hydroxylated.
  • a preferred peptide is H-KJLKL 5 KLK-OH, with all L-amino acids.
  • This oligopeptide is a known antimicrobial [24], neutrophil activator [25] and adjuvant [26] and forms stable complexes with immunostimulatory oligonucleotides to give a good adjuvant.
  • the most preferred mixture of immunostimulatory oligonucleotide and polycationic polymer is the TLR9 agonist known as IC31TM [27-29], which is an adsorptive complex of oligodeoxynucleotide SEQ ID NO: 1 and polycationic oligopeptide SEQ ID NO: 2.
  • the oligonucleotide and oligopeptide can be mixed together at various ratios, but they will generally be mixed with the peptide at a molar excess.
  • the molar excess may be at least 5: 1 e.g. 10: 1 , 15: 1 , 20: 1 , 25: 1, 30; 1 , 35: 1, 40: 1 etc.
  • a molar ratio of about 25: 1 is ideal [30,31 ]. Mixing at this excess ratio can result in formation of insoluble particulate complexes between oligonucleotide and oligopeptide.
  • the complexes can be combined with an oil-in-water emulsion.
  • the oligonucleotide and oligopeptide will typically be mixed under aqueous conditions e.g. a solution of the oligonucleotide can be mixed with a solution of the oligopeptide with a desired ratio.
  • the two solutions may be prepared by dissolving dried (e.g. lyophilised) materials in water or buffer to form stock solutions that can then be mixed.
  • the complexes can be analysed using the methods disclosed in reference 32. They ideally have an average diameter that is larger than the average diameter of oil droplets in the emulsion. Complexes with an average diameter in the range l ⁇ m-20 ⁇ m can be used. In some embodiments there is no overlap between the size distributions of the emulsion and the complexes i.e. the largest droplets in an emulsion are smaller than the smallest complexes (or the largest complexes are smaller than the smallest droplets). In other embodiments, however, the range of droplet and complex diameters may overlap. Poly-arginine and CpG oligodeoxynucleotides similarly form complexes [33].
  • the complexes can be maintained in aqueous suspension e.g. in water or in buffer.
  • Typical buffers for use with the complexes are phosphate buffers (e.g. phosphate-buffered saline), Tris buffers, Tris/sorbitol buffers, borate buffers, succinate buffers, citrate buffers, histidine buffers, etc.
  • complexes may sometimes be lyophilised. Complexes in aqueous suspension can be centrifuged to separate them from bulk medium (e.g. by aspiration, decanting, etc.). These complexes can then be re-suspended in an alternative medium, such as in an oil-in-water emulsion.
  • Adjuvant compositions of the invention will usually be prepared by mixing an oil-in-water emulsion with an oligonucleotide/polymer complex.
  • the emulsion is a liquid and the complexes are typically maintained in liquid form, and so an adjuvant of the invention may be formed by mixing two liquids.
  • one or both of the liquids includes an immunogen so that the mixing provides an immunogenic composition of the invention.
  • neither liquid includes an immunogen, so the mixed product (i.e. the adjuvant composition of the invention) can later be combined with an immunogen to provide an immunogenic composition of the invention.
  • the volume ratio for mixing can vary (e.g.
  • the concentration of components in the two liquids can be selected so that a desired final concentration is achieved after mixing e.g. both may be prepared at 2x strength such that 1 : 1 mixing provides the final desired concentrations.
  • the complexes are not in liquid form (e.g. they have been centrifuged or lyophilised) and they may be combined with (e.g. dissolved in) an emulsion.
  • oligonucleotide and polycationic polymer can be used e.g. any of the concentrations used in references 27, 30 or 31 , or in reference 34.
  • a polycationic oligopeptide can be present at 1 100 ⁇ M, 1000 ⁇ M, 350 ⁇ M, 220 ⁇ M, 200 ⁇ M, 1 10 ⁇ M, 100 ⁇ M, 1 1 ⁇ M, lO ⁇ M, l ⁇ M, 50OnM, 5OnM, etc.
  • An oligonucleotide can be present at 44 nM, 40 nM, 2OnM, 14 nM, 4.4 nM, 4 nM, etc.
  • a polycationic oligopeptide concentration of less than 2000 nM is typical.
  • concentrations in mg/mL in three embodiments of the invention may thus be 0.31 1 & 1.322, or 0.109 & 0.463, or 0.031 and 0.132.
  • squalene-in-water emulsion with an aqueous preparation of IC31 at a 1 : 1 volume ratio can be used to give a composition with the final amounts of components per ml: squalene, 4.9mg; polysorbate 80, 588 ⁇ g; cationic oligopeptide, 500 nMol or 50 nMol; oligonucleotide, 20 nMol or 2 nMol.
  • Adjuvant compositions of the invention usually include components in addition to the emulsion, oligonucleotide and polymer e.g. they typically include one or more pharmaceutically acceptable component. Such components may also be present in immunogenic compositions of the invention, originating either in the adjuvant composition or in another composition. A thorough discussion of such components is available in reference 35.
  • a composition may include a preservative such as thiomersal or 2-phenoxyethanol. It is preferred that the vaccine should be substantially free from (e.g. ⁇ 10 ⁇ g/ml) mercurial material e.g. thiomersal- free. Vaccines containing no mercury are more preferred. Preservative-free vaccines are particularly preferred, ⁇ -tocopherol succinate can be included as an alternative to mercurial compounds in influenza vaccines.
  • a preservative such as thiomersal or 2-phenoxyethanol. It is preferred that the vaccine should be substantially free from (e.g. ⁇ 10 ⁇ g/ml) mercurial material e.g. thiomersal- free. Vaccines containing no mercury are more preferred. Preservative-free vaccines are particularly preferred, ⁇ -tocopherol succinate can be included as an alternative to mercurial compounds in influenza vaccines.
  • a composition may include a physiological salt, such as a sodium salt.
  • a physiological salt such as a sodium salt.
  • Sodium chloride (NaCl) is preferred, which may be present at between 1 and 20 mg/ml.
  • Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, disodium phosphate, and/or magnesium chloride, etc.
  • Compositions may have an osmolality of between 200 mOsm/kg and 400 mOsm/kg, e.g. between 240-360 mOsm/kg, maybe within the range of 280-330 mOsm/kg or 290-310 mOsm/kg.
  • the pH of a composition will generally be between 5.0 and 8.1, and more typically between 6.0 and 8.0 e.g. 6.5 and 7.5, or between 7.0 and 7.8.
  • a composition is preferably sterile.
  • a composition is preferably non-pyrogenic e.g. containing ⁇ 1 EU (endotoxin unit, a standard measure) per dose, and preferably ⁇ 0.1 EU per dose.
  • a composition is preferably gluten free.
  • An immunogenic composition may include material for a single immunisation, or may include material for multiple immunisations (i.e. a 'multidose' kit). The inclusion of a preservative is useful in multidose arrangements. As an alternative (or in addition) to including a preservative in multidose compositions, the compositions may be contained in a container having an aseptic adaptor for removal of material. Compositions will generally be in aqueous form at the point of administration.
  • Vaccines are typically administered in a dosage volume of about 0.5ml, although a half dose (i.e. about 0.25ml) may sometimes be administered e.g. to children.
  • a composition may be administered in a higher dose e.g. about ImI e.g. after mixing two 0.5ml volumes.
  • Immunogens Adjuvant compositions of the invention can be administered to animals in combination with immunogens to induce an immune response.
  • the invention can be used with a wide range of immunogens, for treating or protecting against a wide range of diseases.
  • the immunogen may elicit an immune response that protects against a viral disease (e.g. due to an enveloped or non-enveloped virus), a bacterial disease (e.g. due to a Gram negative or a Gram positive bacterium), a fungal disease, a parasitic disease, an auto-immune disease, or any other disease.
  • the immunogen may also be useful in immunotherapy e.g. for treating a tumour/cancer, Alzheimer's disease, or an addiction.
  • the immunogen may take various forms e.g. a whole organism, an outer-membrane vesicle, a protein, a saccharide, a liposaccharide, a conjugate (e.g. of a carrier and a hapten, or of a carrier and a saccharide or liposaccharide), etc.
  • the immunogen may elicit an immune response against an influenza virus, including influenza A and B viruses.
  • influenza A and B viruses The presence of an oil-in-water emulsion adjuvant (particularly one comprising squalene) has been shown to enhance the strain cross-reactivity of immune responses for seasonal [36] and pandemic [37,38] influenza vaccines.
  • influenza virus immunogen Various forms of influenza virus immunogen are currently available, typically based either on live virus or on inactivated virus. Inactivated vaccines may be based on whole virions, split virions, or on purified surface antigens. Influenza antigens can also be presented in the form of virosomes. Hemagglutinin is the main immunogen in current inactivated vaccines, and vaccine doses are standardised by reference to HA levels, typically measured by SRID. Existing vaccines typically contain about 15 ⁇ g of HA per strain, although lower doses can be used e.g. for children, or in pandemic situations, or when using an adjuvant. Fractional doses such as Vi (i.e.
  • compositions may include between 0.1 and 150 ⁇ g of HA per influenza strain, preferably between 0.1 and 50 ⁇ g e.g. 0.1-20 ⁇ g, 0.1-15 ⁇ g, 0.1-10 ⁇ g, 0.1 -7.5 ⁇ g, O.5-5 ⁇ g, etc.
  • Particular doses include e.g. about 45, about 30, about 15, about 10, about 7.5, about 5, about 3.8, about 1.9, about 1.5, etc. per strain. It is usual to include substantially the same mass of HA for each strain included in the vaccine e.g.
  • HA mass for each strain is within 10% of the mean HA mass per strain, and preferably within 5% of the mean.
  • dosing is measured by median tissue culture infectious dose (TCID50) rather than HA content, and a TCID 50 of between 10 6 and 10 8 (preferably between 10 6 5 -10 7 5 ) per strain is typical.
  • TCID50 median tissue culture infectious dose
  • cell lines that support influenza virus replication may be used. The cell line will typically be of mammalian origin e.g. MDCK.
  • Influenza A virus immunogens may be from any suitable HA subtype strain e.g. Hl , H3, H5, H7, H9 etc., such as a HlNl, H3N2 and/or H5N1 strain.
  • the immunogen may elicit an immune response against a Candida fungus such as C. albicans.
  • the immunogen may be a ⁇ -glucan, which may be conjugated to a carrier protein.
  • the glucan may include ⁇ -1 ,3 and/or ⁇ -1 ,6 linkages.
  • Suitable immunogens include those disclosed in references 41 & 42.
  • the immunogen may elicit an immune response against a Streptococcus bacterium, including S.agalactiae, S.pneumoniae and S.pyogenes.
  • the immunogen may be a capsular saccharide, which may be conjugated to a carrier protein.
  • the saccharide may be from one or more of serotypes Ia, Ib, II, III, and/or V.
  • S.pneumoniae the saccharide may be from one or more of serotypes 1 , 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and/or 23F.
  • polypeptide immunogens may be used to elicit a protective anti-streptococcal immune response.
  • the immunogen may elicit an immune response against a meningococcal bacterium (N. meningitidis).
  • the immunogen may be a capsular saccharide, which may be conjugated to a carrier protein. Capsular saccharides and their conjugates are particularly useful for protecting against meningococcal serogroups A, C, Wl 35 and/or Y.
  • polypeptide immunogens and/or outer membrane vesicles may be used to elicit a protective anti-meningococcal immune response, particularly for use against serogroup B e.g. as disclosed in reference 43.
  • the immunogen may elicit an immune response against a hepatitis virus, such as a hepatitis A virus, a hepatitis B virus and/or a hepatitis C virus.
  • the immunogen may be hepatitis B virus surface antigen (HBsAg).
  • the immunogen may elicit an immune response against a respiratory syncytial virus.
  • Immunogens may be from a group A RSV and/or a group B RSV. Suitable immunogens may comprise the F and/or G glycoproteins or fragments thereof e.g. as disclosed in references 44 & 45.
  • the immunogen may elicit an immune response against a Chlamydia bacterium, including C.trachomatis and C. pneumoniae.
  • Suitable immunogens include those disclosed in references 46-52.
  • the immunogen may elicit an immune response against an Escherichia coli bacterium, including extraintestinal pathogenic strains.
  • Suitable immunogens include those disclosed in references 53-55
  • the immunogen may elicit an immune response against a coronavirus, such as the human SARS coronavirus.
  • Suitable immunogens may comprise the spike glycoprotein.
  • the immunogen may elicit an immune response against a Helicobacter pylori bacterium.
  • Suitable immunogens include CagA [56-59], VacA [60,61], and/or NAP [62-64].
  • the immunogen may elicit an immune response against rabies virus.
  • a suitable immunogen is an inactivated rabies virus [65, RabAvertTM].
  • the immunogen may elicit an immune response against a human papillomavirus.
  • Useful immunogens are Ll capsid proteins, which can assemble to form structures known as virus-like particles (VLPs).
  • VLPs can be produced by recombinant expression of Ll in yeast cells (e.g. in
  • S.cerevisiae or in insect cells ⁇ e.g. in Spodoptera cells, such as S.frngiperda, or in Drosophila cells).
  • plasmid vectors can carry the Ll gene(s); for insect cells, baculovirus vectors can carry the Ll gene(s). More preferably, the composition includes Ll VLPs from both HPV- 16 and HPV- 18 strains. This bivalent combination has been shown to be highly effective [66]. In addition to
  • HPV- 16 and HPV- 18 strains it is also possible to include Ll VLPs from HPV-6 and HPV-1 1 strains.
  • the immunogen may elicit an immune response against a tumour antigen, such as MAGE-I , MAGE-2, MAGE-3 (MAGE- A3), MART-1/Melan A, tyrosinase, gplOO, TRP-2, etc.
  • a tumour antigen such as MAGE-I , MAGE-2, MAGE-3 (MAGE- A3), MART-1/Melan A, tyrosinase, gplOO, TRP-2, etc.
  • the immunogen may elicit an immunotherapeutic response against lung cancer, melanoma, breast cancer, prostate cancer, etc.
  • the immunogen may elicit an immune response against a hapten conjugated to a carrier protein, where the hapten is a drug of abuse [67].
  • hapten is a drug of abuse [67].
  • examples include, but are not limited to, opiates, marijuana, amphetamines, cocaine, barbituates, glutethimide, methyprylon, chloral hydrate, methaqualone, benzodiazepines, LSD, nicotine, anticholinergic drugs, antipsychotic drugs, tryptamine, other psychomimetic drugs, sedatives, phencyclidine, psilocybine, volatile nitrite, and other drugs inducing physical and/or psychological dependence.
  • an immunogenic composition includes a complex of an immunostimulatory oligonucleotide and a polycationic polymer
  • immunogens will usually be adsorbed to the complexes, but this is not required. Thus antigens may, after centrifugation, be associated with the complexes, indicating adsorption.
  • an immunogen is described as being "at least partially adsorbed" to a complex, it is preferred that at least 10% (by weight) of the total amount of that immunogen in the composition is adsorbed e.g. >20%, >30%, >40% or more.
  • an immunogen is described as being "adsorbed" to a complex, it is preferred that at least 50% (by weight) of the total amount of that immunogen in the composition is adsorbed e.g. 50%, 60%, 70%, 80%, 90%, 95%, 98% or more. In some embodiments an immunogen is totally adsorbed i.e. none is detectable in the supernatant after centrifugation to separate complexes from bulk liquid medium, hi other embodiments, though, there is no adsorption.
  • Suitable containers for adjuvant compositions, immunogenic compositions and kit components of the invention include vials, syringes ⁇ e.g. disposable syringes), etc. These containers should be sterile. The containers can be packaged together to form a kit e.g. in the same box.
  • the vial can be made of a glass or plastic material.
  • the vial is preferably sterilized before the composition is added to it.
  • vials are preferably sealed with a latex-free stopper, and the absence of latex in all packaging material is preferred.
  • the vial may include a single dose of vaccine, or it may include more than one dose (a 'multidose' vial) e.g. 10 doses.
  • Useful vials are made of colorless glass. Borosilicate glasses are preferred to soda lime glasses. Vials may have stoppers made of butyl rubber
  • a vial can have a cap ⁇ e.g. a Luer lock) adapted such that a syringe can be inserted into the cap.
  • a vial cap may be located inside a seal or cover, such that the seal or cover has to be removed before the cap can be accessed.
  • a vial may have a cap that permits aseptic removal of its contents, particularly for multidose vials.
  • the syringe may have a needle attached to it. If a needle is not attached, a separate needle may be supplied with the syringe for assembly and use. Such a needle may be sheathed.
  • the plunger in a syringe may have a stopper to prevent the plunger from being accidentally removed during aspiration.
  • the syringe may have a latex rubber cap and/or plunger.
  • Disposable syringes contain a single dose of vaccine.
  • the syringe will generally have a tip cap to seal the tip prior to attachment of a needle, and the tip cap may be made of a butyl rubber. If the syringe and needle are packaged separately then the needle is preferably fitted with a butyl rubber shield.
  • Useful syringes are those marketed under the trade name "Tip-Lok"TM.
  • Containers may be marked to show a half-dose volume e.g. to facilitate delivery to children.
  • a syringe containing a 0.5ml dose may have a mark showing a 0.25ml volume.
  • an individual container may include overfill e.g. of 5-20% by volume.
  • compositions of the invention are suitable for administration to human subjects, and the invention provides a method of raising an immune response in a subject, comprising the step of administering an immunogenic composition of the invention to the subject.
  • the invention also provides a method of raising an immune response in a subject, comprising the step of mixing the contents of the containers of a kit of the invention (or chambers of a syringe) and administering the mixed contents to the subject.
  • the invention also provides composition or kit of the invention for use as a medicament e.g. for use in raising an immune response in a subject.
  • the invention also provides the use of an oil-in-water emulsion, an immunostimulatory oligonucleotide and a polycationic polymer, in the manufacture of a medicament for raising an immune response in a subject. This medicament may be administered in combination with an immunogen.
  • the invention also provides the use of an oil-in-water emulsion, an immunostimulatory oligonucleotide, a polycationic polymer and an immunogen, in the manufacture of a medicament for raising an immune response in a subject.
  • compositions of the invention can be administered in various ways.
  • the usual immunisation route is by intramuscular injection (e.g. into the arm or leg), but other available routes include subcutaneous injection, intranasal, oral, buccal, sublingual, intradermal, transcutaneous, transdermal, etc.
  • Immunogenic compositions prepared according to the invention may be used as vaccines to treat both children and adults.
  • a subject may be less than 1 year old, 1-5 years old, 5-15 years old, 15-55 years old, or at least 55 years old.
  • Preferred subjects for receiving the vaccines are the elderly (e.g. >50 years old, >60 years old, and preferably >65 years), the young (e.g. ⁇ 5 years old), hospitalised subjects, healthcare workers, armed service and military personnel, pregnant women, the chronically ill, immunodeficient subjects, people travelling abroad, etc.
  • IC31TM has been shown to be effective in infant populations [34, 68].
  • the vaccines are not suitable solely for these groups, however, and may be used more generally in a population.
  • Treatment can be by a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunisation schedule and/or in a booster immunisation schedule. In a multiple dose schedule the various doses may be given by the same or different routes e.g. a parenteral prime and mucosal boost, a mucosal prime and parenteral boost, etc. Administration of more than one dose (typically two doses) is particularly useful in immunologically naive subjects. Multiple doses will typically be administered at least 1 week apart (e.g. about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 12 weeks, about 16 weeks, etc.).
  • composition comprising X may consist exclusively of X or may include something additional e.g. X + Y.
  • a process comprising a step of mixing two or more components does not require any specific order of mixing.
  • components can be mixed in any order. Where there are three components then two components can be combined with each other, and then the combination may be combined with the third component, etc.
  • animal (and particularly bovine) materials are used in the culture of cells, they should be obtained from sources that are free from transmissible spongiform encaphalopathies (TSEs), and in particular free from bovine spongiform encephalopathy (BSE). Overall, it is preferred to culture cells in the total absence of animal-derived materials.
  • TSEs transmissible spongiform encaphalopathies
  • BSE bovine spongiform encephalopathy
  • a compound is administered to the body as part of a composition then that compound may alternatively be replaced by a suitable prodrug.
  • a cell substrate is used for reassortment or reverse genetics procedures, or for viral growth, it is preferably one that has been approved for use in human vaccine production e.g. as in Ph Eur general chapter 5.2.3.
  • Figure 1 shows the particle diameters in a mixture of IC31 and MF59.
  • the y-axis shows volume (%) and the x-axis shows particle diameter ( ⁇ m).
  • Figures 2 to 5 show the same analysis for adjuvanted H5N1 antigen at: (2) time zero; (3) 30 minutes; (4) 6 hours; and (5) 24 hours.
  • Figure 6 shows the same analysis for H5N1 antigen adjuvanted with IC31 alone.
  • Figures 7 and 8 show body temperature ( 0 C) over time, from 3 days before infection to 5 days after.
  • a squalene-in-water emulsion, MF59 was prepared as disclosed in Chapter 10 of reference 4.
  • IC31 was prepared in high and low concentrations (10-fold difference) as disclosed in reference 31.
  • Adjuvant combinations were made my mixing MF59 with IC31 hlgh or IC31 low at either a 1 : 1 volume ratio or a 5: 1 volume ratio.
  • the three individual adjuvants (MF59, IC31 high , IC31 low ), and the two mixtures MF59+IC31 low ), have been combined with various immunogens and administered to a variety of mammals to assess their efficacy.
  • IC31 hlgh and IC31 low have been mixed with a MF59-adjuvanted influenza vaccine (FLU ADTM) at a 1 : 1 volume ratio.
  • FLU ADTM MF59-adjuvanted influenza vaccine
  • Influenza antigens were adjuvanted either with MF59 (FLU ADTM), with IC31 (either IC31 hlgh or IC31 l0W ) or with a combination of MF59+IC31 (50 ⁇ L FLU ADTM mixed with equal volume of aqueous IC31 ; both adjuvants mixed at 2x strength to give final Ix after dilution).
  • FLU ADTM MF59
  • IC31 either IC31 hlgh or IC31 l0W
  • a combination of MF59+IC31 50 ⁇ L FLU ADTM mixed with equal volume of aqueous IC31 ; both adjuvants mixed at 2x strength to give final Ix after dilution.
  • Two doses of the various compositions were administered to mice and HI titres were assessed. Based on single samples, titres after the second dose against a HlNl strain of influenza A virus were:
  • HI titres were as follows
  • MF59 and IC31 can enhance HI titres more than either adjuvant alone, particularly for influenza A virus.
  • CD4+ T cells were assessed to determine whether the adjuvants elicited a ThI -type or Th2-type response. Whereas MF59 gave a response that was biased towards a Th2-type response, and IC31 gave a response (at both doses) that was biased towards a ThI -type response (but less strongly biased than MF59), the combined adjuvant was more balanced between ThI -type and Th2-type responses.
  • Hepatitis C virus E1E2 protein was used to immunise mice.
  • the antibody response achieved using MF59+IC31 was similar to the response with MF59 alone, but the highest inhibition of CD81 binding after 3 doses was seen with MF59+IC31 low .
  • Antigen 124 from an extraintestinal pathogenic E.coli strain was adjuvanted with alum, IC31 , MF59 or IC31+MF59. Protection rates were as follows:
  • the antigens from the meningococcus serogroup B vaccine of reference 43 were adjuvanted with MF59, IC31 hlgh , IC31 low or combinations thereof.
  • the vaccine includes three recombinant antigens (AgI, Ag2 & Ag3) and total IgG levels against these were as follows:
  • meningococcal B protein antigens were also combined with conjugated saccharide antigens from serogroups A, C, Wl 35 and Y antigens and were tested with the same adjuvant mixtures.
  • Bactericidal titres against a test strain from each serogroup were as follows:
  • IC31 hlgh was combined with MF59 or with buffer at a 1 : 1 ratio, or with MF59 and buffer at a 1 :0.5:0.5 ratio.
  • MF59 was tested at its normal concentration or at half concentration.
  • the adjuvants were combined with a surface antigen vaccine from a H5N1 strain of influenza virus (A/Vietnam/ 1 193/04). The stability and immunogenicity of the combination was tested.
  • Stability was evaluated by testing pH, osmolality, adsorption, and particle size at time zero and then after 30 minutes or 6 hours of storage at room temperature.
  • the pH was stable in the range 7.23 to 7.26.
  • Osmolality was stable in the range 280-286 m ⁇ sm/kg.
  • the proportion of adsorbed antigen rose from 76% to 80% over 6 hours, having dropped slightly at the 30 minute time point.
  • These figures are similar to control compositions lacking MF59, which had slightly lower osmolality (273-275 m ⁇ sm/kg), a slightly higher pH (7.32-7.35), and a slightly lower proportion of adsorbed antigen (54-68%).
  • FIG. 1 The particle sizes of a IC31 hlgh :MF59 1 : 1 mixture in PBS, at time zero, are shown in Figure 1.
  • the emulsion droplets (mean diameter 161nm) accounted for 46.4% of the volume and IC31 complexes (mean diameter 15.9 ⁇ m) for 53.6%.
  • Figures 2 to 5 show similar analysis but for an adjuvanted vaccine including H5N1 antigen, from time zero through to 24 hours at room temperature:
  • H5N1 antigen in combination with MF59 alone had a mean droplet diameter of 151nm.
  • H5N1 antigen in combination with IC31 hlgh had a mean particle diameter of 38.4 ⁇ m ( Figure 6).
  • Figure 6 the mixing of MF59 and IC31 slightly increases the analysed diameter of MF59 particles (while still permitting sterile filtration) and reduces the diameter of the IC31 complexes. In all cases, though, the mixtures are stable.
  • the ferret ⁇ Mustela putorius furo) model is the preferred animal model to provide evidence of efficacy of candidate pandemic influenza vaccines.
  • adjuvanted vaccines with either 1 ⁇ g of 3.75 ⁇ g of antigen (hemagglutinin dose) were administered to eight groups of ferrets.
  • Ferrets received a priming dose and a boosting dose, and were then challenged with a heterologous H5N1 strain.
  • Figures 7 and 8 show temperatures from two example mice, one in group D ( Figure 7) and one in group E ( Figure 8).
  • HAI titers were assessed against the vaccine strain on days 0, 21 , 42 and 49. Average titers were:
  • S.pyogenes and C. albicans antigens have also been adjuvanted with MF59TM+IC31TM.
  • Vaccine Adjuvants Preparation Methods and Research Protocols (Volume 42 of Methods in

Abstract

La présente invention concerne un adjuvant immunologique comprenant une émulsion d’huile dans l’eau, un oligonucléotide immunostimulateur et un polymère polycationique, caractérisé en ce que l’oligonucléotide et le polymère s’associent idéalement l’un à l’autre pour former un complexe. L’adjuvant peut être combiné avec des immunogènes pour préparer des vaccins.
PCT/IB2009/007111 2008-09-18 2009-09-18 Combinaisons d’adjuvant de vaccin WO2010032138A2 (fr)

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EP2470205A1 (fr) 2009-08-27 2012-07-04 Novartis AG Adjuvant contenant de l'aluminium, un oligonucléotide et une polycation

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CN114010778A (zh) * 2021-10-21 2022-02-08 广州一品红制药有限公司 一种水包油型疫苗佐剂

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